Proteases play an important role in the regulation of biological processes in almost every life form from bacteria to virus to mammals. They perform critical functions in, for example, digestion, blood clotting, apoptosis, activation of immune responses, zymogen activation, viral maturation, protein secretion and protein trafficking.
Proteases have been implicated as the cause of or as contributors to several diseases such as Alzheimer's disease, cystic fibrosis, emphysema, hypertension, tumor invasion and metastasis and viral-associated diseases [e.g., Kim, T. W., et al., (1997) "Alternative Cleavage of Alzheimer-associated Presinilins During Apoptosis by a Caspase-3 Family Protease," Science 277:373-6; Lacana, E. et al., (1997) "Disassociation of Apoptosis and Activation of IL-1 beta-converting Enzyme/Ced-3 Proteases by ALG-2 and the Truncated Alzheimer's gene ALG-3," J. Immunol. 158:5129-35; Birrer, P., (1995) "Proteases and Antiproteases in Cystic Fibrosis: Pathogenic Considerations and Therapeutic Strategies," Respiration 62:25-8; Patel, T., et al., (1996) "The Role of Proteases During Apoptosis," FASEB J. 10:587-97].
Several viral genomes also encode proteases that are important in the viral maturation process. For example, the viral aspartyl protease of the Human Immunodeficiency Virus (HIV) cleaves a HIV polypeptide containing the Gag and Pol polyproteins.
In another example, the hepatitis C virus (HCV) produces a long polypeptide translation product, NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, which is cleaved to produce at least 10 proteins. C, E1 and E2 are putative structural proteins, and the remainder are known as the nonstructural (NS) proteins. One of those proteins is NS3, a 70 kilodalton protein having serine protease activity. It is been shown that the protease activity of NS3 resides exclusively in the N-terminal 180 amino acids of the enzyme. The NS3 protease cleavages at four sites in the nonstructural region of the HCV polypeptide (3/4A, 4A/4B, 4B/5A, and 5A/5B). Another protein, NS4A, has 54 amino acids and has been characterized as a cofactor for the NS3 protease [C. Failla, et al., (1994) J. Virology 68:3753-3760]. The C-terminal 33 amino acids of NS4A are required for cleavage at the 3/4A site and 4B/5A sites and accelerate the rate of cleavage at the 5A/5B site. Several other NS3 serine protease-dependent cleavage site sequences have been identified in various strains of HCV [A. Grakoui, et al., (1993) J. Virology 67:2832-2843 incorporated by reference herein].
The ability to detect viral, cellular, or microorganism protease activity in a quick and simple an assay is important in the biochemical characterization of these proteases, in detecting viral infection, and in the screening and identification of potential inhibitors.
Several protease assays are known in the art. T. A. Smith et al., Proc. Natl. Acad. Sci. USA, 88, pp. 5159-62 (1991); B. Dasmahapatra et al., Proc. Natl. Acad. Sci USA, 89, pp.4159-62 (1992); and M. G. Murray et al., Gene, 134, pp. 123-128 (1993) each describe protease assay systems utilizing the yeast GAL4 protein. Each of these documents describe inserting a protease cleavage site in between the DNA binding domain and the transcriptional activating domain of GAL4. Cleavage of that site by a coexpressed protease renders GAL4 transcriptionally inactive leading to the inability of the transformed yeast to metabolize galactose.
Y. Hirowatari et al., Anal. Biochem., 225, pp. 113-120 (1995) describes an assay to detect HCV protease activity. In this assay, the substrate, HCV protease and a reporter gene are cotransfected into COS cells. The substrate is a fusion protein consisting of (HCV NS2)-(DHFR)-(HCV NS3 cleavage site)-Tax1. The reporter gene is chloramphenicol transferase (CAT) under control of the HTLV-1 long terminal repeat (LTR) and resides in the cell nucleus following expression. The uncleaved substrate is expressed as a membrane-bound protein on the surface of the endoplasmic reticulum due to the HCV NS2 portion. Upon cleavage, the released Tax1 protein translocates to the nucleus and activates CAT expression by binding to the HTLV-1 LTR. Protease activity is determined by measuring CAT activity in a cell lysate.
Each of the assays described above requires simultaneous (1) expression of an active protease and a substrate and (2) transcription of a reporter gene construct. The constitutive nature of these assays can often produce uncontrollable and undesirable effects. These effects may give rise to misleading or inaccurate conclusions regarding the activity of the protease. Thus, there is a need for a sensitive and quantitative protease assay that is inducible or can be readily controlled by the user.